Nanopatterned surfaces and related methods for selective adhesion, sensing and separation

ABSTRACT

The invention is comprised, in part, of a surface that contains more than one component or construct. Such heterogenous surface compositions and configurations, related systems and methods for sensing particle or analyte interaction therewith can selectively and/or differentially interact with a range of particles/analytes, in lieu of specific molecular sensor-analyte interactions for each particle. These interactions of various analytes or particles can differ sufficiently in strength and range between multiple analyte types or particles to effect a separation of analytes or particles mixtures, in a way that requires no sensing or detection. With incorporation of a sensing mechanism, discrimination/detection of different compounds within an analyte mixture can be accomplished.

This application is a divisional of and claims priority benefit fromapplication Ser. No. 12/215,046 filed Jun. 23, 2008 and issued as U.S.Pat. No. 8,117,902 on Feb. 21, 2012, which claimed priority fromprovisional application No. 60/936,861 filed Jun. 22, 2007 and is acontinuation-in-part of application Ser. No. 11/592,454 filed Nov. 3,2006 and issued as U.S. Pat. No. 7,752,931 on Jul. 13, 2010, whichclaimed priority from provisional application Ser. No. 60/732,941 filedNov. 3, 2005—each of which is incorporated herein by reference in itsentirety.

The United States government has certain rights to this inventionpursuant to Grant Nos. CTS-0234166 and CTS-0242647 from the NationalScience Foundation to the University of Massachusetts.

BACKGROUND OF THE INVENTION

Nanometric surface design has been a research focus over the pastdecade, with a variety of concepts and methods developed. Most producefeatures with a size scale greater than 100 nm (e.g., standardphotolithography, microcontact printing and/or with regularityinterference methods). Such methods are labor or equipment intensive.Consider, for instance, the fabrication of a lithographic mask for thefabrication of a complex mold to produce stamps for microcontactprinting. Scribe-type methods (AFM-writing, e-beam lithography, withnanometric resolution) are not only both labor and equipment intensive,but are also substantially limited in terms of surface chemistry. Onlyrecently have interference methods been applied to e-beam lithography,but at the cost of production limited only to regular patterns.

Concurrently, increasing effort has been made to design and fabricatesurfaces with selective properties, for use in conjunction with tissuescaffolds, sensors, smart adhesives, separation media, etc. Selectivityhas been most often achieved, borrowing directly from biology, by thecovalent attachment of biomolecular fragments (both polypeptide andDNA), and the passivation of remaining surface area. The resultingsurfaces bind target molecules, often in micron and slightly submicronpatterns that form the basis for array devices. However, such systems ofthe prior art tend to be limited to detection of specific sensor-analytechemical or immunological interactions.

Most patterned surfaces made by the foregoing techniques are designed tostore information or provide arrays for addressable multi-elementsensing. Wherein such arrays are used for sensor elements, biologicalmolecules (DNA, proteins and antibodies) are placed in various parts ofthe array, thereby imparting specificity to each array element.Informational density and sensitivity can be but are not necessarilypromoted by decreasing sensor size and/or adding more or differentsensor elements. As a result, improved detection continues to presenton-going fabrication challenges.

SUMMARY OF THE INVENTION

In light of the foregoing, it is an object of the present invention toprovide selective separation and sensing compositions and/or articlesand methods for the use and/or assembly, thereby overcoming variousdeficiencies and shortcomings of the prior art, including those outlinedabove. It would be understood by those skilled in the art that one ormore aspects of this invention can meet certain objectives, while one ormore other aspects can meet certain other objectives. Each objective maynot apply equally, in all its respects, to every aspect of thisinvention. As such, the following objects can be viewed in thealternative with respect to any one aspect of this invention.

It is an object of the present invention to provide heterogeneoussurface compositions, relative amounts of components A and B, surfacelengthscale for distribution of A on B, lengthscale for the size of Adomains and/or chemical nature of A and B, for selective interaction ofparticles or analytes exposed thereto, such heterogeneity as can beconsidered in terms of such selectivity at least in part related toparticle/analyte size, and/or local curvature.

It can be another object of the present invention to provide such acomposition, surface and/or related method to selectively distinguishparticles/analytes in a size range from less than about 50 nanometers togreater than about 10 microns, without limitation as to the chemicalcharacteristics of any such particle/analyte.

It can be another object of the present invention to provide any suchcomposition, surface, system and/or related method using spatialdimension, spatial configuration and/or density of corresponding surfacecomponents to effect or control selective adhesion, separation ordetection.

It can be another object of this invention to provide such a surfacerandomly configured, with nanoscale features, so as to avoid costlypatterning technologies of the prior art.

It can be another object of the present invention to provide aheterogeneous or patterned surface, for use with a related system ormethod, robust to pH, temperature and other environmental factors, andwhich selectively interacts with target particles or analytes over thoseoutside a predetermined size range or physical/chemical profilecharacteristic.

It can be another object of certain embodiments of this invention toprovide such selective interaction without opposed charges on thesurface (e.g. plus) and surface component (e.g., negative) with respectto an analyte or particle exposed thereto.

It can be another object of the present invention to provide aheterogeneous surface, system or related method, selectively and/ordifferentially interactive with a range of particles/analytes, each withits own recognition pattern, in lieu of a specific sensor-analytechemical interaction for each particle.

Other objects, features, benefits and advantages of the presentinvention will be apparent from this summary and the followingdescriptions of certain embodiments, and will be readily apparent tothose skilled in the art having knowledge of various separation/sensingtechniques. Such objects, features, benefits and advantages will beapparent from the above as taken into conjunction with the accompanyingexamples, data, figures and all reasonable inferences to be drawntherefrom, alone or with consideration of the references incorporatedherein.

In part, this invention can comprise a method for particle sensingand/or of using a spatial surface configuration for selective particleinteraction. Such a method can comprise providing a heterogeneoussurface comprising a surface member and a plurality of componentsthereon, such components spaced about said surface and having an averagesurface density, the heterogeneity comprising different interactions ofthe surface member and the spaced components with a particle or analyteexposed thereto; exposing a particle or analyte to the heterogeneoussurface; and sensing different interactions of the particle or analytewith the heterogeneous surface, selective for such a particle. Withoutlimitation, such particle interaction can comprise adhesion and/orseparation.

While various interactions, e.g., physical or chemical, can be utilizedin conjunction with this method, in certain embodiments, theheterogeneity on the surface member can comprise one or more of a rangeof electrostatic and/or non-electrostatic interactions with a particleor analyte (optionally, of a net charge or comprising another physicalor chemical characteristic), whereby the surface member and the spacedcomponents thereon can provide variation in surface electrostatic chargeand/or other non-electrostatic character (e.g., without limitation,hydrophobicity, hydrogen-bonding capability, etc.). In particular, thespaced components can, themselves, have a surface charge density ornumber, or comprise a variation in surface charge and/or hydrophobiccharacter, at least partially sufficient for selective particleinteraction; and ionic strength can be further varied, as determined, toalter or modify selectivity. Regardless, such surface components canalso comprise a dimension (e.g., without limitation, up to about 50 nmand an average spatial density at least partially sufficient forselective particle interaction. Such density can be varied as may berequired for enhanced selectivity of a specific particle/analyte.Accordingly, charge density, intracomponent charge and/or hydrophobiccharacter variation and/or spatial density can be utilized, modifiedand/or altered for selective interaction with a first particle of afirst dimension, such selectivity over a second particle with a second,different radial dimension and/or localized surface radius of curvature.Particle displacement can provide such a surface for subsequent orrepetitious exposure and sensing.

Without limitation, particle/analyte sensing can comprise a rate ofadhesion to the heterogeneous surface and one or more other interactivesignatures including but not limited to rolling, skipping and arrest,such interactions as would be understood by those skilled in the art.Accordingly, sensing can comprise adhesion and/or one or more signaturesor a sequence of signatures for a specific particle/analyte interaction,whereby such a method can be used for selective sensing upon exposure ofa particle mixture to the heterogeneous surface. In certain embodiments,as illustrated below and approaching an adhesion threshold for aparticular heterogeneous surface, such interaction can provide forseparation of a particle/analyte from a medium and/or from a pluralityof different particles/analyte types exposed to such a surface. Removalof adhered/interacted and/or separated particles permits use of thesurface for subsequent exposure and sensing.

In part, this invention can also be directed to a system for selectiveparticle or analyte sensing. Such a system can comprise a heterogeneoussurface comprising a surface member and a plurality of componentsthereon, such components spaced about and having a surface density, withthe heterogeneity comprising different interactions of the surfacemember and of the spaced components with a particle/analyte exposedthereto. As discussed elsewhere herein, various surface heterogeneitiesand different interactions can be utilized; however, in certainembodiments, competing electrostatic interactions, or a combination ofelectrostatic and non-electrostatic interactions with a particle of netcharge can be provided using a surface member having a charge oppositeto the surface components thereon.

In certain embodiments a surface member can have a net negative charge,and a spaced component can have a net positive charge. Such anembodiment is available through deposition of one of severalcommercially available cationic polymers onto a silica surface at levelsbelow those corresponding to surface saturation (e.g., withoutlimitation, up to about 50% of saturation). As discussed elsewhereherein, component spacing can be optimized to provide an interactiveand/or adhesion threshold of such a heterogeneous surface for a specificparticle/analyte. In certain non-limiting embodiments, components spacedfrom about 15 nanometers to about 60 nanometers can be used to sensemicron-dimensioned particles. In certain other embodiments, componentsspaced from about 15 nanometers to about 30 nanometers for selectiveparticle sensing. Such spacing or density can be varied, separately orin conjunction with the size and/or charge density of the spacedcomponents. Accordingly, as described more fully below, such a systemcan be designed and used to selectively sense particles of a givendimension or chemical/physical characteristic.

In certain other embodiments, a surface member can have a net negativecharge, and a spaced component, as can comprise a protein material, cancomprise portion(s) of net positive charge and/or portion(s) of netnegative charge, as can be present with hydrophilic and/or hydrophobicportion(s). Such an embodiment is available through deposition of one ormore of several commercially available proteins onto a silica surface atlevels below those corresponding to surface saturation (e.g., withoutlimitation, up to about 50% of saturation). As discussed elsewhereherein, component spacing can be optimized to provide an interactivethreshold of such a heterogeneous surface for a specificparticle/analyte. In certain non-limiting embodiments, such a surfaceprotein component can comprise a fibrinogen, and such components can bespaced on average from about 15 nanometers to about 60 nanometers tosense micron-dimensioned particles. In certain other embodiments,components can be spaced from about 20 nanometers to about 35 nanometersfor selective particle sensing. Such spacing or density can be varied,separately or in conjunction with the size and/or charge density of thespaced components. Accordingly, as described more fully below, such asystem can be designed and used to selectively sense particles of agiven dimension or chemical/physical characteristic.

In part, this invention can also be directed to a method for determiningparticle signature. Such a method can comprise providing a heterogeneoussurface comprising a surface member and a plurality of componentsthereon, such components spaced about and having an average density onthe surface member, with the surface heterogeneity comprisingdifferential interactions of the surface member and spaced componentswith an exposed particle and selectively interactive therewith; exposingone or more known types of particles to the heterogeneous surface;sensing interactions of the known particles with the heterogeneoussurface; and determining one or more signature interactions, inrecognition of each known particle. Such signature(s), once determined,can be used for purposes of comparison with a signature of an unknownparticle/surface interaction to identify the unknown particle.

As mentioned above and discussed more fully below, surface heterogeneitycan give rise to various different or competitive surface interactionswith an exposed particle. Likewise, in certain embodiments, a surfacemember and spaced components thereon can comprise domains of opposedcharges for different electrostatic interaction with a particle having anet charge. Likewise, in certain other embodiments, a surface member andspaced components thereon can comprise domains of charge (or, e.g.,without limitation, chemical) variation for different electrostaticand/or non-electrostatic interaction with a particle having a net chargeor physical-chemical affinity. Charge density, surface spacing, spatialdensity and/or domain size can be varied for selective or optimalinteraction of such a heterogeneous surface with a particle/analyte of agiven dimension or curvature. Such interactive signatures can compriseadhesion, arrest, rolling, skipping and/or other interactive signaturesrecognized by those skilled in the art, whereby a interactive patterncan ascertained, in recognition of the known particle. As such, exposureof a second or unknown particle and comparison with a recognizedinteractive signature can be used to determine or assess identity of asecond/unknown particle.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C. Schematic illustrations (not to scale) of a charge-basedheterogeneous surface and spatially varying interactions with threetypes of potential analyte particles in A, B, and C. On theheterogeneous surface of such non-limiting embodiments, the positivedomains can be on the order of 10 nm in diameter and their averagespacing can vary from zero to over 100 nm, and particle size can rangefrom less than about 200 nm to greater than several microns.

FIG. 2. Schematic illustration of a positively charged domain. Therepresentation on the left shows the adsorbed polycation (single chain)while that on the right emphasizes the charge distribution and downplaysthe backbone conformation.

FIG. 3. Limiting low-coverage particle (460 nm silica spheres) adhesionkinetic data at 3 different positive domain (pDMAMEA) densities (10%,14%, 100%) on a planar collector (heterogeneous surface). Ionic strengthis 0.005M. F is the adherent mass of silica particles.

FIG. 4. Adhesion rates of 460 nm silica particles as a function ofdomain density for I=0.005M.

FIG. 5. Schematic illustration portraying a Zone of Influence and itsradius, R_(zi). Here a sphere of radius R_(p) contacts the patchy planarsurface, during conditions give a Debye length, κ⁻¹. R_(zi) iscalculated according to right triangles: R_(zi) ²+Rp²=(R_(p)+κ⁻¹)²

FIG. 6. Effect of ionic strength on adhesion threshold for 1 μmparticles.

FIGS. 7A-B. (A) Activation energy follows from colloidal potential (B)Activation energy follows from additive colloidal and hydrodynamicpotentials.

FIGS. 8A-C. One method of measuring particle adhesion rates to surfaces(heterogeneous and otherwise) is Total Internal Reflectance Fluorescence(TIRF). Shown here are TIRF data for the adhesion of 460 nmRhodamine-core silica spheres having, negative surfaces, onto a planarsurface made positive by a saturated layer of pDMAEMA. (A) Continuedparticle deposition for several hours until the particle deposition rateis substantially reduced by surface crowding. The bulk particleconcentration is 0.1 wt % and the wall shear rate is 39 s⁻¹. (B) Initialdeposition kinetics at the same wall shear rate and different bulkparticle concentrations. The inset shows explicit dependence of thedeposition rate on the particle concentration. (C) Control runs showingthe particle adhesion rate onto a surface made uniformly positive bypDMAEMA saturation is independent of bulk ionic strength in the range0.005-0.026M, and also that there is no particle adhesion onto baresilica for this batch of 460 nm spheres. Here, buffer is reinjectedafter 20 minutes of particle deposition.

FIG. 9. Optical micrographs of 460 nm particles adhering to fullypositive collectors, for runs like that in FIG. 3A, but interrupted atdifferent times for optical microscopy: (A) 3 minutes (B) 10 minutes and(C) 500 minutes. Note that the dynamic signatures and recognition oftarget particles/analyte by heterogeneous surfaces can be observed atparticle levels much lower than exemplified here with opticalmicroscopy.

FIGS. 10A-B. Comparative adhesion rates of silica particles ontoheterogeneously electrostatic surfaces, as a function of particle sizeand domain density. (A) ionic strength is 5 mM. (B) ionic strength is 26mM.

FIGS. 11A-B. (A) optical micrograph of the charge-wise heterogeneoussurface from case i of FIG. 10A, after exposure to a flowing suspensioncontaining fluorescent-core 460 nm silica particles (65 wt %) and 1 μmnon-fluorescent silica particles (35 wt %) and aggregates of the 460 nmparticles; and (B) optical micrograph of the charge-wise heterogeneoussurface from case ii control of FIG. 10A. While the suspension that waspassed over surface ii did contain some aggregates of the 460 nmparticles, there were fewer aggregates in this suspension than thatwhich was flowed over surface i.

FIG. 12. Graphic representation of data showing reversible particle (1μm silica) adhesion. More than 99% of the particles (I=0.026M) can beremoved or displaced from a 10% positive patch/component surface.

FIG. 13. A schematic illustration of a (bovine, Type IV) fibrinogenprotein.

FIG. 14. Zeta potentials of 0.5 and 1 μm silica particles containingdifferent amounts of adsorbed bovine serum fibrinogen.

FIG. 15. Comparative adhesion rates of silica particles ontoheterogeneous surfaces, as a function of fibrinogen nanoconstruct loads.Capture rates of 0.5 and 1 μm silica particles on silica surfaces, fromflowing pH 7.4 buffer at the ionic strengths indicated. The wall shearrate is 24 s⁻¹. Adhesion thresholds for the different data sets areindicated by the corresponding arrows.

FIG. 16. Capture Rates of 1 μm silica particles on surfaces containingdifferent amounts of Fibrinogen (x-axis). Data for 3 different flowrates are shown. The pH is 7.4 and the ionic strength is 26 mM.

FIG. 17. Velocity distributions comparing velocities of rollingparticles on fibrinogen-saturated and pDMAEMA-saturated surfaces.

FIG. 18. Silica versus staph capture rates on silica surfaces that havebeen saturated with adsorbed fibrinogen (4.5 mg/m²), at pH 7.4 and anionic strength of 26 mM. The hollow symbols are the staph bacteria whilethe solid symbols are the silica particles.

BRIEF DESCRIPTION OF CERTAIN EMBODIMENTS Description of RepresentativeHeterogeneous Surfaces.

Without limitation to any one theory or mode of operation, variousaspects and features relating to certain embodiments of this inventioncan be considered in conjunction with FIGS. 1A-C. In FIG. 1B, a negativemicron-scale sphere interacts with a negative surface carrying patchesof multiple positive charges, where the patches are substantiallysmaller than the sphere. At any position, the sphere experiences bothattractions and repulsions at different points on its surface. Thesphere is currently in an attractive well, and translation of the sphereover the surface results in a height-dependent spatially varyingpotential. A different sized sphere would experience a differentproportion of attractions and repulsions such that the heterogeneoussurface will attract some particle sizes better than others. In theexperimental scenario of FIG. 1B, a relatively smooth negatively chargedparticle interacts with an electrostatically heterogeneous surfacepresenting attractive (positive) domains on an otherwise repulsive(negative) surface, where the domains are small relative to the particlesize. Lateral variations in surface charge (the heterogeneous domains)attract or repel the sphere, depending on its position in the x, y, andz directions. Thus the approaching sphere experiences a potential energylandscape. In FIG. 1B, the particle experiences this landscape bytranslating across the surface. This situation is similar to the moreclassical pattern-recognition situation, in FIG. 1A where, with apattern on both sides of the interface, attractions and repulsionsresult from particle translation and rotation. The surface of themicron-scale sphere illustrated in FIG. 1A contains multi-chargedpositive domains. Depending on the size and spacing of the positivedomains on the sphere relative to those on the surface, patternrecognition and adhesion, with various dynamic signatures, may or maynot be achieved, thus producing selectivity. In the example shown, thesize and spacing of the domains on the sphere are similar to those onthe heterogeneous planar surface giving rise to the possibility ofadhesion. The sphere is currently in a repulsive maximum and translationor rotation will cause the potential to vary. A slight rotation ortranslation from the current position would move the sphere toward alocal minimum, producing adhesion. If the patch distribution on thesphere were out of registry with that on the plane, a more complicatedpotential with weaker attractions would result. Likewise in FIG. 1C, asimilar affect may be observed for a particle with surface roughness ofa particular lengthscale, but a relatively uniform charge distribution,in this case negative. Here the analyte is not a sphere, but anaggregate of smaller primary spheres, carrying only negative charge. Inaddition to the lengthscale of the overall analyte particle, there is alengthscale of the small primary spheres making up the aggregate. Inthis example, the small sphere lengthscale is in registry with thelengthscale implicit in the heterogeneous surface, giving rise toattractions. A different size primary particle in the aggregate wouldbreak this registry and reduce attractions and adhesion.

As demonstrated below, tuning the interfacial features of FIG. 1B allowsmanipulation of adhesion dynamics, including an adhesion threshold.Further, the adhesion dynamics of particles such as those in FIG. 1C(where large particles are aggregates of smaller primary particles,imparting a surface roughness lengthscale) are distinct from that ofsmooth particles with the same overall size and surface charge densityas that in FIG. 1B. Adhesion or sensing occurs with pattern featuredensity above the threshold, but not below it. Utilizing this invention,a selective behavior is observed for the system in FIG. 1B, one moretypically associated with the classical pattern recognition scenario inFIG. 1A. Selectivity is also observed for the system in FIG. 1C.

Implementing such features, this invention can be demonstrated using theinteractions between micron-scale silica particles and planar surfacescontaining randomly-arranged physiadsorbed nanoconstruct adhesivepatches of varied density (with the remaining surface area beingelectrostatically and/or otherwise relatively repulsive towards theparticles). Rather than AFM, colloidal probe, or surface forces methods,particle deposition from gently flowing solution was employed to assessthe interactions from a practical perspective and to demonstrate patternrecognition-like, selective features of the interactions. Adhesion rateswere observed as a function of the domain density, and a certain rangeof surface conditions were found to promote adhesion, much like theconcept of interfacial pattern recognition.

Representative of one or more embodiments of this invention, anddemonstrating but one utility thereof, heterogeneous planar surfaceswere made by adsorbing controlled amounts of a cationic polyelectrolyte,poly(dimethylaminoethyl methacrylate), pDMAEMA, onto the negativesurface of an acid washed microscope slide. At high coveragesapproaching saturation, adsorbed polymer layers typically form a densecarpet over a surface, but at lower coverages, individual chains can bedistinguished by the right techniques. In this example, the individualpolymer chains comprise the positive domains. The domains roughly can beenvisioned as round with order 10 nm diameter, but flat to the surfaceand randomly placed, based on a large bank of data in the literature.(See, Shin, Y.; Roberts, J. E.; Santore, M. M. Macromolecules 2002, 35,4090-4095; Shin, Y. W.; Roberts, J. E.; Santore, M. M. Journal ofColloid and Interface Science 2002, 247, 220-230; Hansupalak, N.;Santore, M. M. Macromolecules 2004, 37, 1621-1629; Hansupalak, N.;Santore, M. M. Langmuir 2003, 19, 7423-7426.) Roundish domains (ratherthan substantially elongated or extended chains) would be expected inthe limit where small amounts (isolated chains) of pDMAEMA adsorb to thesilica surface, because under typical adsorption conditions (whichinclude an ionic strength of 0.026 M, and gentle shearing flow) pDMAEMAis a coil rather than an extended chain in free solution. The fastadsorption rate and the strong (irreversible) adsorption energeticsminimize the potential for substantial reconformations of segments afterthey contact the surface, so that the free coil conformation should bereflected at least roughly by the adsorbed footprint or domain size.(See, Irurzun, I. M.; Matteo, C. L. Macromolecular Theory andSimulations 2001, 10, 237-243; Hoogeveen, N. G.; Stuart, M. A. C.;Fleer, G. J. Journal of Colloid and Interface Science 1996, 182,133-145; Hansupalak, N.; Santore, M. M. Macromolecules 2004, 37,1621-1629.)

The particular pDMAEMA utilized had a molecular weight of 31,300 (200monomers) and a polydispersity of 1.1. A hydrodynamic coil radius,R_(H), of 4 5 nm measured by dynamic light scattering approximates theradius of gyration, R_(g) to first order for these coils in solution.The adsorbed chain footprint is expected to be similar, with the patchdiameter approximated by the free coil end-end distance, 6^(1/2)R_(g),11 nm. Since pDMAEMA is a weak polyelectrolyte its protonation ispH-dependent: At the pH of 6.1 in this study, pDMAEMA is 70% protonatedin free solution, but because of the relatively dense spacing of theseunderlying backbone charges, counterion condensation reduces the netbackbone charge to a spacing approaching the Bjerrum length of 0.7 nm,or 74 net positive charges/chain. (See, Shin, Y. W.; Roberts, J. E.;Santore, M. M. Journal of Colloid and Interface Science 2002, 247,220-230; Shin, Y. W.; Roberts, J. E.; Santore, M. Journal of Colloid andInterface Science 2001, 244, 190-199.)

The individually adsorbed DMAEMA chains would be expected to producerelatively flat (order 1 nm thick) domains, per conventional wisdom forthe adsorption of substantially-charged polyelectrolytes. Indeed, NMRstudies, at conditions relevant to this study, found adsorbed pDMAEMAchains to lie flat to the surface: At pH's of 7 and below, saturatedlayers (with coverages of 0.45 mg/m²) are 80% trains (the part of thechain that contacts the surface), and no more than 20% loops and tails.At coverages below 0.1 mg/m², where interesting adhesion kinetics ofsilica particles were found in the current work, the adsorbed polymer is100% trains within detectable limits. This means that the polymerdomains are relatively flat to the surface, extending only the thicknessof the backbone (order 1 nm) into solution. Domain arrangement on thecollector surfaces is expected to be random, especially at low domaincoverages when chain spacing exceeds the Debye length. Aggregation orclustering of adsorbed chains is improbable since positive backbonecharge causes interchain repulsion. Additionally, no aggregation isobserved in bulk at pH 6 over a broad range of ionic strength.

A concern in using adsorbed polymer as a component of random surfacepatterning is that it stays adsorbed to the underlying planar surface.In this work pDMAEMA was employed at pH 6.1 where shown that it does notdesorb or exchange with material from solution over a period of days.While salt often facilitates polyelectrolyte desorption, in this systemat pH 6 or 7 it has almost no effect on the adsorbed amount, presumablydue to dense backbone charging. Because pDMAEMA chains are so immobileagainst desorption or self exchange with other pDMAEMA chains, a lowlateral mobility of these chains would also be expected—when encounteredby colloidal particles—to promote steady position. In fact, controlsemploying fluorescent pDMAEMA demonstrated complete domain retention onexposure to particles.

Another feature of the heterogeneous surface of this invention is itselectrostatic landscape. It is well known that saturated layers ofpolyelectrolytes with sufficient backbone charge can reverse theunderlying substrate charge. This behavior can enable multilayerstructures containing negatively- and positively-chargedpolyelectrolytes. Indeed saturated pDMAEMA layers on silica, in a pHrange of the sort described herein, are known to completely reverse theunderlying silica charge. While the interesting adhesion thresholdregion in this invention corresponds to sufficiently low pDMAEMAcoverages to produce isolated adsorbed coils, the region in the vicinityof each coil would be expected to be positively charged. Indeed, theconcept that single chains can contribute electrostatic domains is wellestablished in the cationic flocculant literature. When adsorbing chainsare too short to induce bridging flocculation, addition of small amountsof densely positively charged polymer to negatively charged colloids caninduce flocculation by electrostatic attraction of the positive domainsadsorbed on one sphere to the negative bare surface of a collidingparticle.

In the literature, the electrophoretic mobility of pDMAEMA adsorbed onsilica spheres (a model approximating planar surfaces herein) was linearin the adsorbed amount of pDMAEMA, over the full range of pDMAEMAcoverages from bare silica to saturated pDMAEMA layers. (Shin, Y. W.;Roberts, J. E.; Santore, M. M. Journal of Colloid and Interface Science2002, 247, 220-230.) That observation indicates that each adsorbingchain, from the first chain to adsorb onto a bare silica surface to thelast to incorporate into a saturated layer, brings the same net chargeto the interface. In the dilute surface limit of just a few pDMAEMAchains on a relatively bare silica surface, positive charge should belocalized in the vicinity of the adsorbed chains. Measurements of theelectrophoretic mobility, and titrations of the charge in the polymersolutions and colloidal dispersions led to calculations of the chargeassociated with each adsorbed chain. At pH 6, adsorbing pDMAEMA releasessodium ions from silica's double layer and promotes further ionizationof surface silanols, which locally increases the underlying negativecharge on the silica. Even with this charge regulation, however, eachadsorbing pDMAEMA chain brings +28 charges to the surface (through thecombined processes of adsorption, counterion release, and silica chargeregulation). (The electrokinetically measurable charge is lower due tocounterions present.) By comparison, the silica bare silica surfacecharge density is 0.16-ve charges/nm² at pH 6. Accordingly, a surfacecan be considered by the scenario in FIG. 2, which includes ˜11 nmdomains with 28 positive charges each, in a sea of negative charge withaverage density −0.16/nm², and the domain density can be varied to giveinteresting adhesion results with particles in solution.

Demonstration of Adhesion Rate Control.

As a reference point in characterizing the adhesion rates of colloidalparticles, the deposition of silica particles was examined on microscopeslides carrying saturated (full) layers of pDMAEMA, with a coverage of0.45 mg/m². These polymer layers were adsorbed at pH 6.1 I=0.026M and,at these conditions, the underlying silica surface charge is completelyreversed. In other words, the negative silica surface is made positive.Such positively charged surfaces should be strongly adhesive towardsnegatively charged particles in solution. As described below and in thefollowing examples, silica spheres exhibited transport-limited particleadhesion to full pDMAEMA layers, confirming the absence of an energybarrier between the negative particles and a relatively uniform positiveplanar surface. The confirmation of the transport-limited adhesion rateonto positive surfaces carrying full pDMAEMA layers demonstrates thequantitative accuracy of the adhesion rate measurement and confirms ourunderstanding of principle in the uniform surface (particle-surfaceattractions) regime.

FIG. 3 shows initial adhesion kinetics of 460 nm silica particles onplanar surfaces carrying different amounts of positive domains: 10%,14%, and 100%. Percent-domain loading is arbitrarily defined relative tothe saturated pDMAEMA layer of 0.45 mg/m². Since the interfacial chargeis linear in the amount of pDMAEMA deposited, and since at pH 6 thesaturated pDMAEMA surface almost exactly overcompensates the underlyingsilica charge, 50% domains corresponds to a planar surface with net zerocharge, even though the substantial charge is distributed betweenpolymer-rich and silica-rich regions of the surface. The “percent”description of domain loading is not defined in terms of surface areathough the result is actually close in this example. At a saturationcoverage of 0.45 mg/m², the apparent footprint of each chain is 87 nm²(corresponding to a disk with diameter 10.5 nm) This footprint diameteris in good agreement with the 11 nm diameter light scattering estimatefrom the free coil size. While in reality adsorbed polymer layers aretypically thought of as entangled carpets of chains, as the surface ismade more dilute, individual coils should ultimately becomedistinguishable (by the right experimental probes). Results areespecially interesting in the dilute limit where the question ofadsorbed chain identity is not blurred by the possibility of chainoverlap on the surface.

In FIG. 3, particle adhesion onto a surface containing 100% domains (asaturated pDMAEMA layer) proceeds at the transport limited rate per theprevious discussion, indicating fundamentally fast underlying adhesionkinetics between the spheres and the collector. With 14% and 10%domains, however, the deposition rate becomes increasingly slower,indicating a reduction in the fundamental sticking rate of particles asthey approach the interface. The finite adhesion rate constantsassociated with these heterogeneous surfaces can be thought to reflectan energy barrier resulting from competing attractive and repulsiveforces acting on a single particle, and the fact that particlesapproaching the interface in a locally negative region of the collectorwill have a greater probability of rejection than those more directlyapproaching the positive domains.

FIG. 4 summarizes the data in FIG. 3 and similar runs for a bulksolution concentration of 0.1 wt % particles and an ionic strength of0.005M. The x-axis is linear in the pDMAEMA loading on the collector,represented as “% domains”; and a second x-axis shows the averagespacing between the centers of the pDMAEMA coils (domains). The lefty-axis shows the particle adhesion rate while the right y-axistranslates this to a set of adhesion rate constants. The right y-axisapplies towards the threshold in the surface-dominated regime. Withfaster particle capture rates, the observed rate constant becomesrepresentative of transport conditions. Several interesting featuresappear, starting on the right hand side of the graph (high or densedomain loading on the collector) where the transport limited particledeposition rate persists for a substantial range of relatively highpositive domain densities: Even when the surface has substantialnegative regions, particles approaching the planar collector can quicklyfind positive regions of the surface where they adhere. This is the caseat 50% domain density, where transport-limited deposition occurs eventhough the surface is net neutral, and down to about 25% domain loadingwhere the surface carries a substantial net negative charge and theaverage center-center domain spacing is about 19 nm. In this largetransport-limited regime of particle deposition, from 25-100% domainloading, adhesion of the 460 nm silica particles was observed to beirreversible. That is, like the data in FIG. 3, the particles were notdisplaced from the surface during buffer flow subsequent to theirinitial deposition. The particle adhesion rate only becomes noticeablyslow (relative to the transport limit) for average positive domainspacings of 20 nm or more.

As the average spacing between the positive domains is increased and asthe surface becomes increasingly net negative, the particle adhesionrate decreases. Ultimately the particle adhesion rate approaches zero,at a finite value of the average domain spacing, near 28 nm. It isnotable that the sloping branch of the data in FIG. 4 does not insectthe origin, but rather defines a threshold interfacial condition foradhesion. From another perspective, the fact that the data miss theorigin suggest that one domain alone on a negative surface is notsufficient to trap and hold a particle (otherwise there would be finiteparticle accumulations and measurable rates for all patch loadingsgreater than zero). Hence, adhesion of particles in the methods andsystems of this invention is believed to rely on spatial fluctuations inthe domain placement on the surface: particles tend to selectivelyadhere to regions of the surface containing a higher than averagedensity of positive domains. Accordingly, the nanometric features ofthis invention can be used for recognition of analytes or componentsranging from about 10 to about 1,000 times larger than the domain size.

Another interesting observation for the data in FIG. 4 is that in thelimit of slow particle adhesion (for average positive domain densitiesof about 15% and below), the particle adhesion was reversible. That is,for the most negative surfaces to which particles adhered, they could bewashed off in a matter of minutes simply by flowing pH 6 buffer (withI=0.005M). This observation is consistent with the concept of domaindensity-dependent adhesion and de-adhesion rate constants for theparticles: Particle adhesion onto more positive surfaces ischaracterized by a relatively large adhesion rate constant andapparently irreversible adhesion (slow deadhesion rates). The morenegative the surface, the slower the forward adhesion reaction and themore accessible the deadhesion kinetics. Reference is also made to FIG.12, a graphic presentation of data showing reversible particle adhesionover time.

Considering the concept of a threshold surface lengthscale, FIG. 5illustrates another factor which can determine whether or not a particlewill adhere to a heterogeneous surface: e.g., the lateral area on acollector surface with which a particle interacts as it approaches asurface. FIG. 5 can be viewed as an illustration to estimate the maximumsize of this “zone of influence” (on the collector), based—in thiscase—on electrostatics. Parallel geometric arguments would, forinstance, hold with steric repulsions. Consistent with this particularembodiment, a particle is placed in hard contact with the surface, andthe zone of influence is defined as the area on the planar surface thatis intersected by an imaginary shell around the particle, correspondingto the Debye length; that is, a “zone of influence” of radiusR_(zi)=[(Rp+κ⁻¹)²−R_(p) ²]^(1/2). For a particle size of 460 nm and aDebye length of 4 nm (corresponding to I=0.005M buffer) R_(zi)=43 nm.Alternately, include the Debye layer near the planar surface in thecalculation: If the intersection of the Debye layers of the sphere andthe planar surface are considered, R_(zi)=62 nm. The two estimates arethe same within an order 1 multiplicative constant.

Regardless of how the zone of influence is defined, its radiusapproaches the average spacing between the domains at the adhesionthreshold (R_(zi)=40-60 nm, average patch spacing 30 nm), furthersupporting the idea that spatial fluctuations in the domain arrangementgovern particle adhesion. If one considers a circle of radius R_(zi) (43nm as an example) on the collector, one calculates an average of 5.5domains in this zone at the adhesion threshold of 11% domains. Eachdomain carries a net 28 positive charges (this calculation includes theeffects of counterion release on adsorption and of charge regulation,the localized increase in the underlying silica in the region of thepatch). Despite the presence of these 7 domains in a 5800 nm² zone ofinfluence, the average surface character is substantially net negative.In fact, 25 domains would be required to completely neutralize the zoneof influence. If one assumes that particle adhesion requires at least anet neutral region of the surface, then at the adhesion threshold,particles adhere to localized regions with more than 3-4 times theaverage positive domain density. As the radius of the “zone ofinfluence” approaches the average inter-patch spacing (at the adhesionthreshold), such fluctuations become more probable, compared to thesituation, for instance, when the zone of influence is much larger orsmaller than the average domain distance.

As discussed above, nanometer scale surface features can be tuned tomanipulate the adhesion of micron-scale objects. An appropriatelengthscale to be considered is not necessarily the particle size butthe size of the interactive zone between the sphere and the plate,compared with the scale of the features on the planar surface. Thus, thelocal curvature can dominate the overall particle size, as alluded to inFIG. 1C. For the electrostatic system studied here, these lengthscalesare well defined and approach each other at conditions the adhesionthreshold. The presence of an adhesion threshold, gives rise to use ofthis invention for selective adhesive behavior, as is illustrated belowin a specific example.

Reduction of solution ionic strength to increase the Debye length andhence R_(zi) finds the threshold shifted to higher average densities ofpositive domains, as shown in FIG. 6. (A similar behavior is observedfor particle size variations, based on the definition of R_(zi)). Largerzones of influence more nearly reflect the average collector properties(net negative charge) while small zones of influence are sensitive topositive concentration fluctuations, the basis for size selectivity evenwithout patterns on the particles. (Patterned particles, such a those inFIG. 1B, would experience an even more dramatic selectivity.) Theinfluence of ionic strength confirms the concept of an electrostaticzone of influence and that the modified surfaces of this invention candiscriminate particles by size or charge density. FIG. 6 highlights theregion of selective adhesion for particle size, via R_(zi), tunablethrough ionic strength.

Selectivity.

With reference to examples 5a-b, below, charge-wise heterogeneoussurfaces were generated with positive domain densities corresponding tocase i and case ii in FIG. 10A. The two surfaces were then exposed toflowing suspensions containing 65 wt % (with respect to silica content)460 nm fluorescent-core plain shell silica particles and 35 wt % (withrespect to silica content) 1 μm non-fluorescent (pure) silica. The 460nm particles had been centrifuged and resuspended by mixing andultrasonication, as part of their synthesis/purification procedure and,as a result of incomplete resuspension, the 460 nm particle portion ofthe mixture contained a small amount of aggregates of the 460 nm primaryparticles. This included some doublets and triplets, but also aggregatesseveral microns in size. In the supply container which fed the flowchamber, these aggregates were the largest particles present and settledto towards the bottom of the container faster than the primary 460 nmand 1 μm particles. When the tube which fed the flow chamber wasimmersed deep into the supply container, these aggregates were pumpedinto the flow cell along with the primary particles. As a result, theseaggregates were present in the bulk solution for both case i and case iiruns. However, due to the position of the tube, there were greaternumbers of aggregates present in the bulk solution of the case i run.

The micrograph of FIG. 11A shows the surface for case i after exposureto the mixture containing 460 nm fluorescent spheres, 1 μmnon-fluorescent particles, and aggregates of the 460 nm particles. Hereonly the 460 nm particles and aggregates thereof adhered to the surface,while 1 μm particles were rejected, indicating the case i surface wasselective for 460 nm spheres and larger objects having local curvaturecorresponding to 460 nm. The 1 μm particles which were rejected by thecase i surface were hydrodynamically and mass-moment smaller than theaggregates of the 460 nm particles, yet they were rejected by surfacebecause of their larger radius of curvature.

The micrograph of FIG. 11B shows the control run for this study, usingthe case ii surface. This surface captured all types of particles (460nm and 1 μm primary particles, and aggregates of the 460 nm particles)injected into the flowcell. While FIG. 11B is a light micrograph whichshows all the particles on the surface, switching to fluorescent lightmade the 1 μm spheres invisible, proof that both sized particlesadhered, as expected.

These results illustrate the selectivity of the charge-wiseheterogeneous surfaces, favoring adhesion of smaller objects or complexobjects with small radii of curvature, while rejecting large smoothspheres possessing greater radii of curvature.

Dynamic Signatures.

In addition to the ability to control the adhesion rates and adhesionreversibility of particles using heterogeneous surfaces, preliminaryoptical microscopy studies revealed interesting dynamic signatures.Substantially positive collector surfaces produced irreversible particleadhesion; however, moderate domain spacing yielded reversible adhesionwith single exponential detachment kinetics and τ=50-500 s, shown herefor conditions where the ionic strength is 0.026 M. In the reversibleregime, particles rolled or skipped as they first encountered thesurface, reminiscent of neutrophil (a type of white bloodcell)interactions with the endothelium. Adhesion at an injury occurs byrolling, mediated by the forming and breaking of selectin-carbohydratebonds. Arrest follows as integrins bind more tightly. Without selectinsto first reduce the neutrophil velocity, integrins do not bind. Byanalogy, dynamic signatures associated with this invention can compriseany one or a combination of adhesion, rolling, arrest and the like.Indeed, with controlled chemistries and distributions of features,sensor surfaces can distinguish the approaching surfaces of bacteriahaving size and charge characteristics comparable to the silica spheresutilized herein, based on the signature of their interfacial motion,leaving an active (not spent) sensing area.

Detection Schemes.

Detection can employ TIRF, optical reflectometry, or microscopy toevaluate particle adhesion and, in the reversible regime, particledetachment. All characterization for particles and surfaces can beconducted as needed by available methods described herein or asotherwise known in the art. In addition to material variations (particleand collector chemistries, domain dimensions and density, chargedensity) that affect the interplay of colloidal forces, systematic flowrate variations can produce adhesion signatures such as rolling,skipping, and arrest. Particle motion can be quantified with opticalmicroscopy using a horizontal flow chamber that avoids the domination ofinterfacial forces by gravity. However, different signatures arepossible by tipping the flow chamber over such that gravity adds to ordetracts from the particle surface attractions. Further, studies canemploy existing steady shear flow chambers that provide a uniform wallshear rate and an additional flow cell geometry, with an increasingchamber cross section, to spatially vary shear in a single experiment,probing critical conditions for particle arrest. Experiments and theorytarget the limit of low particle coverage, relevant to high sensitivity,early detection, and adhesion fundamentals.

Adhesion can be analyzed as described herein, where TIRF andreflectivity signal was proportional to the instantaneous amount ofparticles adhering to the detector. In the low particle coverage limit,with strong (irreversible) adhesion onto highly attractive surfaces, theforward adhesion rate law can be assessed when the mass transport rateis sufficiently large. In the reversible regime (with less attractivesurfaces), the removal (in flow) of previously adhering particles canreveal the detachment rate law. The particle capture rate laws, likelyfirst order, can depend on the particle concentration while the adhesionrate constants k_(on) and k_(off) can depend on a system's“chemical-force” parameters (collector surface chemistry and patchiness,choice of particle, ionic strength and flow rate). The latter controlthe colloidal interaction of the particle with the surface landscape.Maxima in these potentials should act as activation energies, E_(a), perFIG. 7 and establish the rate constants via Kramer's rate theory:k=ωe^(Ea/kT). The attempt frequency, ω, can, in principle, be determinedby temperature variations; however, temperature is a thermodynamicparameter (e.g. affecting solvent quality) not merely a dynamic one.Therefore, the ionic strength, which varies the electrostatics in waysthat are known, can systematically vary the potential barrier,decoupling ω and E_(a). External forces such as hydrodynamics add tocolloidal potentials to reduce the barrier height and producevelocity-dependent rate constants, a treatment to be used, per FIG. 7B.

Regarding molecular rate constants for individual particles or surfacecomponents, particle rolling and skipping occurs, direct measurement ofk_(on) and k_(off) is complicated, because bonds simultaneously form andbreak at the leading and trailing edges of the zone of influence. Inthis regime, k_(off) can be measured in detachment experiments thatfollow deposition of a small amount of particles onto a surface in apreconditioning step. Microscopy can confirm the extent to whichparticles can be removed or roll in different shear flows. K_(on) canfollow from measurements of the steady-state (equilibrium) particledensity on the surface. With weak binding, this equilibrium particlecoverage should be quite low. Measurements of rolling velocities (anddistributions thereof) and any rolling or skipping prior to final arrestcan be measured, and interpreted in the context of a model whichincludes the probability of particles encountering favorable patches attheir leading edges.

Representative of one or more other embodiments of this invention, anddemonstrating but one utility thereof, heterogeneous planar surfaceswere made by adsorbing controlled amounts of the protein bovine serumfibrinogen as a nanoconstruct, onto the negative surface of an acidwashed microscope slide. Fibrinogen comprises two symmetrical halves,each containing three polypeptide chains (Aα, Bβ, γ). These sixpolypeptides form fibrinogen's structure, which is comprised of acentral e-domain and two terminal d-domains. The αC arms are extensionsof the Aα peptides from the d-domains and are negatively charged. TheN-terminus of all six polypeptides resides in the central e-domain,giving this domain an overall positive charge which allows electrostaticbinding of the αC arms. The cleavage of the end sequences of the Aα andBβ peptides by thrombin in the e-domain results in the formation offibrin, an activated state of fibrinogen able to associate andpolymerize. (See, also, Feng, et al., In Proteins Interfaces II:Fundamentals and Applications; American Chemical Society: Washington,D.C. 1994; Vol. 602, pp. 66-79. and, as can relate to one or moreembodiments of this invention, reference is made to the schematicillustration of FIG. 13, below as provided in Feng et al.)

Referring to FIG. 14, zeta potential was determined for a series ofbatches of silica particles onto which different amounts of bovine serumfibrinogen have been adsorbed. The silica particles are representativeof the planar collecting surfaces in the invention, and the pH and ionicstrength have also been chosen to match the experimental conditions ofthe flowing particle studies, like those in FIG. 15, below. Rather thanusing a planar surface, the zeta potential of silica particles wasmeasured because it is experimentally the easiest way to assess theaverage electrostatic surface charge. Accordingly, particles wereconstructed that resemble a planar surface: a silica surface containingsmall amounts of an adhesive entity, e.g., fibrinogen.

FIG. 14 shows that, over the full range of experimental conditions,silica particles with different amounts of fibrinogen are net negative,allowing conclusion that the present planar silica collecting surfacesare net negative, despite the different amounts of fibrinogen adsorbed.By comparison to the data with pDMAEMA, fibrinogen-containing silicasurfaces are always net negative at the pHs and ionic strengthsemployed. As discussed above, the pDMAEMA-containing silica surfaceswere negative below about 0.225 mg/m² pDMAEMA, for the particularmolecular weight employed. (As a point of interest in that embodiment,above about 0.225 mg/m² pDMAMEA the collecting surfaces become netpositively charged.) More generally, fibrinogen is net negativelycharged and, while it has hydrophilic peptide domains, it has asubstantially hydrophobic character. Fibrinogen is roughly 4.5×4.5×47nanometers in free solution, and may become slightly larger in width(the small dimensions), when absorbed to silica. The physical forcesthat bind fibrinogen to a planar silica surface are sufficiently strongto ensure its immobilization. The origin of attractions between adsorbedfibrinogen (on silica) approaching silica particles could be the smallamounts of localized positive charge, hydrophobicity, hydrogen-bonding,or alteration of the water structure of the fibrinogen. There is areduced, but still negative zeta potential on silica coated withfibrinogen, suggesting that the outer surface of the adsorbed layer offibrinogen still possesses net negative charge. This is the case evenwhen the surface is fully coated with fibrinogen. (See, Tosanco andSantore, Langmuir, 2006, 22, pp 2588-2597, the entirety of which isincorporated herein by reference.) Use of physiological pH 7.4 makes asilica surface somewhat more negative (by 20-25%) in charge than thesilica at pH 6, as compared with and described in conjunction with theaforementioned cationic polymer embodiments.

The present fibrinogen-based nanopatterned/nanotextured surfaces wereprepared as described in the examples, with controlled amounts offibrinogen from flowing solution, buffered at pH 7.4. The particularsamples below discussed in the context of FIG. 15, were fabricatedemploying laminar slit shearing cells, as described in example 12. Thesurfaces contain less than the surface-saturated amount of protein. Suchsurfaces are generated by flowing protein for a limited time and thenreinjecting buffer into the cell, in much the same fashion as thesurfaces generated in the aforementioned, incorporated application.However, it is not necessary to use flow cells to fabricate suchsurfaces. The flow cells do increase quantitative precision inscientific studies, and facilitate perfectly planar surfaces. Thenanoconstructs could also be adsorbed to the surface of arbitrarilyshaped objects or even fibers and column packings by immersing theseobjects into a solution or suspension of nanoconstructs for a controlledtime period, and then rinsing.

A series of data demonstrating the adhesion of fibrinogen-bearing silicasurfaces towards silica particles is shown in FIG. 15. In this study,each data point results from an experiment in which a suspension ofmonodisperse spherical silica particles (1000 ppm) was flowed over afibrinogen-bearing silica surface, at pH 7.4, a wall shear rate of 24s⁻¹, with the ionic strength and particle sizes indicated. Near-BrewsterReflectometry was employed to determine the adhesion or capture rates ofthe particles on the y-axis, in the limit of low coverages of silicaparticles on these collecting surfaces. In FIG. 15, the x-axis shows theamount of fibrinogen on each of a series of silica surfaces, representedas mass fibrinogen per unit area of surface, and also summarizing theaverage spacing between absorbed fibrinogen molecules. The y-axis showsthe rate of silica particle adhesion to these surfaces, from flowingbuffer solution, at pH 7.4 and two ionic strengths.

Towards the right side of the graph of FIG. 15, all 4 data sets (for 2particles sizes, each at two ionic strengths) approach thetransport-limited adhesion rates, determined by the flow cell geometryand by particle size. As smaller particles diffuse more quickly thanlarger ones, the adhesion rate of the 500 nm particles is greater thanthat for the 1 μm particles. As the amount of adsorbed fibrinogen on thecollecting surface is decreased (moving towards the left side of thegraph), the particle adhesion rates ultimately become dependent on thefibrinogen loading on the collector—differently for each of the twoparticles sizes, and also dependent on ionic strength. Considerfibrinogen surface adsorption decreased by about 50 percent (e.g., from2.4 to about 1.2 mg/m²). In this range, particle capture rate isconstant, but surfaces with less fibrinogen capture particles moreslowly.

Ultimately, towards the left side of the graph (FIG. 15), the data setsall exhibit finite x-intercepts rather than passing through the origin.These x-intercepts are adhesion thresholds, surface compositions belowwhich no particle capture occurs. The presence of an adhesion thresholdindicates that more than one fibrinogen molecule is necessary to captureeach silica particle--indicating that the arrangement of the adsorbedfibrinogen molecules can be an important consideration in determiningadhesion. In light of previously demonstrated random arrangement offibrinogen adsorbed on silica (Toscano and Santore, Langmuir 2006,supra), the current data support the lengthscale of the surface spatialfluctuations in fibrinogen arrangement as a consideration in theadhesion dynamics of the silica particles. This data, demonstrate thatsurfaces with random arrangements of nanoconstructs can control theadhesion rates of approaching particles. The data also demonstrate thatthere are some collector surface compositions which adhere small 500 nmparticles but reject 1 μm particles. The effect of ionic strength is dueto the charged nature of the silica particles and the negative charge onthe main part of the planar silica surface, giving rise to a backgroundrepulsion that becomes screened at higher ionic strengths: Approachingparticles experience forces from a large are of the collecting surfaceat low ionic strengths, making it more difficult for clusters offibrinogen to impose attractions.

The data sets in FIG. 15 demonstrates that with a system where theattractions between the surface nanoconstructs and the approachingparticles are other than purely electrostatic, the adhesion rate ofparticles can be controlled, and that such surfaces can discriminateobjects by size or curvature. As can be interpreted from FIG. 15,different sized particles (i.e., those with different hydrodynamic sizeand curvatures) are captured at different rates, as can depend on theionic strength of the solution. A feature of FIG. 15 is the adhesionthresholds: their presence indicates that surfaces of such an embodiment(i.e., choice of conditions on the x-axis) will capture small particles(or sharply curved particles) and completely reject bigger particles orthose with smoother curvature. The separation is discriminatory. Thatis, choice of surface embodiment can be used to completely reject thelarger, smoother particles. These data are similar to and consistentwith those from a purely electrostatic system. Comparison is made, forinstance, with FIGS. 10A and 10B. Likewise, such data support use of thepresent invention to determine various adhesion signatures (skipping,rolling, sliding, arrest, etc.) as additional means by which particles,interactive with a heterogeneous surface, can be distinguished.

As demonstrated herein, surfaces with nanoscale features can be used todistinguish objects, whether from a suspension or otherwise in a fluidmedium. While some sensors and assays of the prior art immobilize DNAfragments or receptors and ligands on surfaces to individually acting asadhesion agents at the molecular level, the present approach does notrely on the one-to-one adhesion behavior of fibrinogen or any otherprotein with a particular analyte. That is, the present invention doesnot require molecular-level recognition to discriminate micron-scale andsubmicron-scale objects. The biological nature of fibrinogen isimmaterial, as this molecule is merely a representative choice, among arange of other available proteins or proteinaceous materials, todemonstrate the principle of control over the adhesion of micron-scaleobjects with a nano-patterned surface. For instance, varioussurface-adsorbed proteins can be used with comparable effect. Withoutlimitation, other net positively-charged proteins include lysozyme andchymotripsin. Net negatively-charged proteins include, withoutlimitation, various immunoglobulins and avidins. Other proteinaceousmaterials, whether net negatively- or positively-charged, can beselected from and are as would be known to those skilled in the art madeaware of this invention. Regardless, such heterogeneous surfaces andadhesion thresholds can be specifically designed for selective adhesivemanipulation (e.g., without limitation, separation) of particles oranalytes, including biological particles/analytes such as, withoutlimitation, bacteria, viruses and cellular material.

EXAMPLES OF THE INVENTION

The following non-limiting examples and data illustrate various aspectsand features relating to the systems and/or methods of the presentinvention, including the use of heterogeneous surfaces having variousspatial components/arrays as are available through the fabricationtechniques described herein. In comparison with the prior art, thepresent systems and methods provide results and data which aresurprising, unexpected and contrary thereto. While the utility of thisinvention is illustrated through the use of several such surfaces andparticles interacting therewith, it will be understood by those skilledin the art that comparable results are obtainable with various otherpatterned heterogeneous surfaces and interacting particles, as arecommensurate with the scope of this invention.

PDMAEMA with a molecular weight of 31,300 and a polydispersity of 1.1was a gift from DuPont, supplied in a THF solution. Rotary evaporationwas used to replace the original THF solvent with water. Final samplepurity was confirmed via proton NMR spectroscopy. A handful of controlruns tracking the presence (and lack of removal) of positively chargedpDMAEMA surface domains employed a lightly rhodamine-b-tagged version ofpDMAEMA. The labeling procedure has been described previously, and inthe current study, the labeling density was 1 label/13 chains. Of note,rhodamine was found to be noninvasive in mobility studies of pDMAEMA onsilica. (See, Hansupalak, N.; Santore, M. M. Macromolecules 2004, 37,1621-1629.)

Bovine plasma fibrinogen, type IV, was purchased from Sigma and was 95%clottable. In experiments requiring fluorescent traces, fluoresceinisothiocyanate (Sigma) was covalently attached to fibrinogen by reactionat room temperature in carbonate buffer (0.004 M Na₂CO₃ and 0.046 MNaHCO₃) for several hours, according to established procedures. Freefluorescein and other potential contaminants (including proteinaggregates) were removed from the protein solution using size exclusionchromatography with a BioGel P-6 polyacrylamide gel column (Biorad). Thecolumn eluent was phosphate buffer (0.008 M Na₂HPO₄ and 0.002 MKH₂—PO₄), such that the purified, labeled product was at pH 7.4 ratherthan the pH 9 corresponding to the reaction solution. Buffer salts werepurchased from Fisher Scientific. The extent of fluorescein labeling wasmeasured with absorbance at 494 nm and, for different labeling batches,was between 0.7 and 1.3 per fibrinogen molecule.

The planar substrates were the surfaces of microscope slides (FisherScientific, Pittsburgh, Pa.) which had been treated with sulfuric acidand rinsed in a sealed flow cell to produce a pure silica surface, freefrom contamination by airborne organics. Previous XPS studies confirmedthe removal of sodium, calcium, and other salts from the region near thesurface, leaving the exposed silica layer. This silica layer, measuredoptically, is on the order of 10 nm thick, with a refractive index of1.49.

pH 6.1 (±0.05) buffer solutions (with I=0.026M) were made using 0.0234 MKH₂PO₄ and adding a very small amount of 0.000267 M NaOH. This solutionwas diluted as necessary to achieve the dilute buffer concentration(I=0.005M) in this work. Negligible effect of buffer concentration on pHwas found in the range (0.005-0.1M), and neither acid nor base wereadded for further pH adjustment.

Example 1a

Charge-wise-heterogeneous planar surfaces were generated by adsorbingvaried amounts of pDMAEMA onto these silica surfaces from a 20 ppmflowing pH 6.1 buffered solution (at ionic strength, I=0.026M), using alaminar slit flow cell with a 10×40 mm slit machined into a black Teflonblock and sealed against the microscope slide substrate using an o-ring.Continuous gentle flow (with wall shear rates in the range 10-50 s⁻¹)maintained a constant bulk solution concentration and defined themass-transport conditions. Saturated (completely positive andovercompensating the underlying silica substrate) surfaces weregenerated by allowing the pDMAEMA solution to flow over the surface fora few minutes longer than needed to saturate the surface. To generateheterogeneous surfaces, adsorption from gentle shearing flow was allowedto proceed only for a few seconds (a time which was systematicallyvaried to tune the domain density) before the flow was switched back topure buffer. Adsorbed pDMAEMA chains were aged in this buffer for 10minutes before exposure to silica particles in adhesion tests, a timeperiod found to yield fully relaxed pDMAEMA layers (eliminating anypotential history dependence).

Example 1b

With reference to the preceding example, various other cationicpolyelectrolytes can be physically adsorbed to a negatively chargedsurface, silica or otherwise, to provide other heterogeneous surfaces inaccordance with this invention. Likewise, such charged components canundergo further chemistry for covalent attachment to such a surfacemember. Alternatively, spaced components comprising one or moreavailable anionic polyelectrolytes can be employed on a positive surfacemember (e.g., a cationic self-assembled monolayer) for a reversed chargeconfiguration. While planar silica slides were used as a negativesurface member/substrate, the systems and methods of this invention canbe employed with other oxide surfaces or, likewise, with non-planarsurface/substrate configurations, for instance, the surfaces of fibers.For instance, a positively charged surface member can be constructedfrom a coating or monolayer of a material such asaminopropyltriethoxysilane coupled or applied to a suitable substrate.Negatively charged spaced components can comprise an anionicpolyelectrolyte such as but not limited to polyacrylic acid. Theeffectiveness of such a heterogeneous surface can be demonstrated usingcationic latex to simulate sensing a positively charged particle. Othersuch heterogeneous surface constructions are as would be understood byone skilled in the art made aware of this invention, such surfaces ascan be employed according to the methods and systems described herein.

Example 2

Monodisperse silica particles serve as an analyte, to exemplify theworkings of the invention. Microscopy and optical reflectometrydetection methods sense all silica particles utilized, while totalinternal reflectance fluorescence and fluorescence microscopy requirefluorescent silica particles. In the case of fluorescent particles, theywere synthesized such that a fluorescent dye resides in the core of theparticle. The outer shell of the silica particle contains no dye andpresents a pure silica surface (with negative charge) to the surroundingfluid. The fluorescent cores of some samples of particles allowed themto be distinguished from other, non-fluorescent particles that wereadded to the analyte suspension in some of the examples below.

To synthesize colloidal silica particles with fluorescent cores, amodified Stober process was used. (See, Vanblaaderen, A.; Vrij, A.Langmuir 1992, 8, 2921-2931; Stober, W.; Fink, A.; Bohn, E. Journal ofColloid and Interface Science 1968, 26, 62-&.) Tetraethoxysilane (TEOS,Sigma) was freshly distilled before the synthesis to remove all theaggregates; (3-aminopropyl)triethoxysilane (APTS, Aldrich) was usedwithout purification. Ethanol (200 proof, VWR), and ammonium hydroxide(Sigma-Aldrich, 25%, analytical reagent quality) were filtered through0.2 μm filters to remove dust prior the synthesis. In the first step,which produced a “dye precursor,” Rhodamine B isothiocyanate (Sigma),was covalently attached to APTS by mixing the two in anhydrous ethanol(with 25% excess of APTS) and allowing them to react under the nitrogenflow for at least 12 hours. In the second step, seed formation,freshly-made dye precursor was added to an ammonia-ethanol mixture alongwith TEOS and stirred gently for 10 hours. Four steps of growing werethen done, adding the same amount of TEOS and allowing mixture to reactfor at least 8 hours for each step, to progressively place shells ofuntagged silica over the core. After that, particles were gentlycentrifuged to remove supernatant with unreacted dye and APTS and washedwith ethanol 2 times. Next, rinsed particles were re-suspended in anethanol-ammonia mixture and another 4 growth steps were performed.Particles were then washed 4 times with ethanol, and 7 times with water.Particle size was characterized using SEM and DLS. Both methods gavesimilar results—a diameter of about 460 nm and a low polydispersity of1-2%.

Example 3

Total internal reflectance fluorescence was used to measure the adhesionrates of the fluorescent-core silica spheres and, in control studiesemploying fluorescent pDMAEMA, polymer adsorption. A TIRF cell inside aSpex Fluorolog II spectrometer was employed, as previously described inthe literature. Excitation light was at 553 nm and emissions weremeasured at 573 nm. An evanescent penetration depth near 100 nm, in ourinstrument, easily excites labels on a nanometer-thin layer of adsorbedpolymer, but it also is highly effective to excite the cores of 460 nmsilica particles. Though an evanescent wave with a decay length of 100nm would appear not to be able to reach into the core of aninterfacially adhesive half-micron silica sphere, evanescent light cantraverse a thin gap of low refractive index to tunnel into a higherrefractive index medium. The evanescent wave tunnels into the sphere andscatters, exciting the rhodamine containing core. Indeed, fluorescencesignal from interfacially adhesive rhodamine-core silica particles wassome of the largest in our experience with TIRF.

Example 4a

The data of this example demonstrate several principles supporting theutility of this invention; that is, various particle/surfaceinteractions.

Non-fluorescent layers of pDMAEMA were deposited prior to theintroduction of fluorescent silica particles at time zero. Separatecontrol studies with fluorescent pDMAEMA were conducted to confirm thatno polymer was removed during interaction of the layer with particlesfrom solution. FIG. 8A presents TIRF data for the adhesion of the 460 nmfluorescent-core silica spheres on the fully adhesive fully positive,attractive (saturated pDMAEMA) surface. With a bulk concentration of 0.1wt % and a wall shear rate of 39 s⁻¹, particle adhesion is initiallylinear in time, but levels off within 5 hours. Towards the end of therun, pure pH 6.1 buffer was injected and the particles were not rinsedoff the surface, indicating irreversible (for practical purposes)adhesion. Particles could not be removed even when the ionic strengthwas raised to 1M or DI water was introduced. FIG. 8B further exploresthe initial particle adsorption kinetics onto saturated pDMAEMA layers,focusing on the influence of particle concentration. In each of theseruns, buffer is injected at the arrows and the lack of signal dropindicates particle retention on these surfaces. In the inset it is clearthat the initial adhesion rate increases in linear proportion to thebulk particle concentration, one of the signatures of transport-limiteddeposition. FIG. 8C demonstrates that the initial (limiting low particlecoverage) particle deposition rate is independent of ionic strength (inthe range 0.005-0.026M). (This was also true of the initial pDMAEMAcoverage, which is not shown.) Also in FIG. 8C, another controlexperiment is shown: The silica particles do not adhere to acid etchedslides not carrying pDMAEMA. This confirms the repulsive interactionbetween a bare collector surface and the silica particles. (This resultis non-trivial as several batches of silica particles from commercialsources failed this test.)

Example 4b

FIG. 9 presents optical micrographs of surfaces generated in runs likethat in FIG. 8A. After in-situ cleaning and rinsing of the microscopeslide in the flow cell, a saturated pDMAEMA layer was deposited from abulk solution of 85 ppm and a wall shear rate of 39s⁻¹ (at pH 6.1 and anionic strength of 0.026M). After 10 minutes, the layer was exposed topure buffer for another 10 minutes. Next, a 0.1 wt % dispersion of 460nm silica spheres was passed over the surface for the times indicated inparts A-C of FIG. 9, after which the flow was switched back to buffer toremove the free particles from the bulk solution. During this finalbuffer rinse, no particles were removed from the surface, as evidencedby a lack of fluorescence signal drop. At this point, the TIRF cell wascarefully dismantled and the microscope slide dried and moved to anoptical microscope to record the image at the central observation point(corresponding to the mass transfer conditions in the active TIRF area).FIG. 9 therefore shows the adherent particles, for different times inthe run of FIG. 8A.

In FIGS. 9A-C, it is clear that the silica particle coverage increasessubstantially with increased particle deposition time, as expected. Alsoof note is the random arrangement of particle deposition and lack ofcolloidal ordering, though some doublets are present. At long times (inFIG. 9C) there is no evidence for multilayer formation and, indeed, nonewould be expected since the flow cell is oriented vertically, so thatgravity does not aid deposition. At short times (i.e., FIG. 9A), it waspossible to count the adherent silica particles. For the run at 3minutes, we counted a total of 236 particles per 40×50 μm square (40×50μm² is the full field, only close-ups are shown in FIG. 9), whichcorresponds to a silica particle coverage of 13.2 mg/m².

Example 5a

A comparison was made of the adhesion rates of monodisperse 460 nm and1000 nanometer silica particles onto several series of heterogeneoussurfaces, whose domain density is varied as shown on the x-axis. SeeFIGS. 10A-B. (100% domain density corresponds to a saturated uniformlayer of pDMAEMA which carries substantial positive (attractive)charge). In general, on the right side of each of these graphs where thecollecting surfaces are predominantly pDMAEMA, the particles adherequickly since the collecting surface charge is net positive while theparticles are negative. 50% patches corresponds to a net neutrality ofthe collector surface, though locally there are positively andnegatively charged regions. With less pDMAEMA positive domain loading onthe surface (greater spacing between the domains), the adhesion rategenerally slows and in all cases the adhesion rate goes to zero at apositive x-intercept.

As discussed above, that the data do not pass through the graphic originsuggests that more than one adhesive domain or surface element is neededto trap each particle on the surface. The x-intercept for each data setcorresponds to an average surface domain density that acts as athreshold below which no adhesion occurs. Note that this threshold (andthe specific particle adhesion rates) depends on particle size andcharge density, a basis for particle selectivity. The threshold belowwhich no adhesion occurs is consistent with the concept of patternrecognition illustrated in FIG. 1.

To demonstrate selectivity, two heterogeneous component surfaces,designated by the lines “case i” and “case ii” in FIG. 10A, were exposedto a mixture of 460 nm and 1000 nm particles (65 and 35 wt %,respectively), which also contained aggregates of the 460 nm particles.Line i passes through the data curve in FIG. 10A for the 460 nanometerparticles with a finite adsorption rate of 0.75 mg/m²/min, but theadhesion rate of the 1000 nm particles for the same conditions on thesame surface is zero. Therefore only 460 nm particles and aggregates of460 nm particles should adhere. Line ii corresponds to a surface with agreater density of surface domains, acting as a control. Here there arefinite, albeit different adhesion rates of both particles.

Example 5b

Optical microscopic images (FIGS. 11A and 11B) were also obtained forsurfaces exposed to a mixture of particles as illustrated in FIG. 10A atlines (i) and (ii). For (i), after a half-hour exposure to the particlemixture, only the smaller particles (460 nm) and aggregates thereofadhered to the subject surface—demonstrating selectivity of this surfacefor the smaller particles or species of small local curvature. (See FIG.11A.). In contrast, for (ii), both the smaller particles and aggregatesof the smaller particles and larger (1000 nm) particles adhere. (SeeFIG. 11B.) There are substantial numbers of the larger particles on thissurface. Such results were confirmed using fluorescence microscopy: thesmaller particles contained fluorescent cores and were apparent; thelarger particles were not fluorescent, but appeared as dark spots in thefluorescence image. The surface of case ii indiscriminately capturedboth the larger and smaller particles, again demonstrating selectivitycan be engineered by controlling the average spacing of surfacecomponents on a collecting surface member. Reference is made to thepreceding discussion regarding the presence and generation of particles(e.g., 460 nm), aggregates (e.g., 1000 nm), aggregates of the smallerparticles (e.g., greater than 460 nm) and mixtures thereof.

Example 6

For interfacially adherent objects which act as Brownian diffusers infree solution, the Leveque equation can describe the transport-limitedaccumulation of these objects on a collector, dF/dt, from the steadystate solution to the convection-diffusion equation in a slit shearcell:

$\begin{matrix}{\frac{\Gamma}{t} = {\frac{1}{{\Gamma \left( {4/3} \right)}9^{1/3}}\left( \frac{\gamma}{DL} \right)^{1/3}{{DC}_{o}.}}} & (1)\end{matrix}$

Here, C_(o) is the bulk solution concentration, γ is the wall shearrate, D is the bulk solution diffusivity, and L is the distance from theentrance to the point of observation in the cell. On the right side ofequation 1, (and only there), Γ(4/3) is the gamma function evaluated foran argument of 4/3. Equation 1 was successfully applied to describe theadsorption kinetics of polymers and proteins in shear cells. Applying itto 460 nm silica particles (with a Stokes-Einstein-diffusivity of1.062×10⁻⁸ cm²/s), a deposition rate, dΓ/dt, of 0.0712 mg/m²s would beanticipated for the linear regime of FIG. 8A. At 3 minutes thiscorresponds to a coverage of 12.8 mg/m² which is in excellent agreement(less than 5% error) with the particles counted in the micrograph ofFIG. 9A, 13.2 mg/m². Such results indicate that the adhesion rate of 460nm particles on the positive collector is the transport-limited ratecorresponding to a lack of energy barrier between the particle and thesurface. The observations also confirm that the 460 nm particles are toosmall to exhibit hydrodynamic lift at the interface, which would tend toretard their adhesion. Thus the micrographs confirm that the maximumadhesion rate is obtained for these negative silica particles on theuniformly adhesive (positive) surface. With the calculation in Example6, FIG. 9A provides an independent calibration for the TIRF instrumentin runs such as those in FIG. 8.

Example 7

Using the transport limited regime as a measure of coverage, theultimate coverage level on the plateau of FIG. 8A is approximately 0.42g/m², which is substantially less than the coverages of 0.53 and 0.61g/m² which correspond to square and hexagonal packing of 460 nm silicaspheres on a planar surface. The coverages corresponding to the initialdeposition kinetics are far below these levels, indicating that theinitial adhesion kinetics of particles on the heterogeneous surfacescharacterize the bare (single particle) interactions between theparticles and the surfaces, per FIG. 1.

Example 8

Consistent with the foregoing, other tests employed ml-quantities offluid containing 10⁸ spheres/ml (˜10⁻⁴ volume fraction), a dynamicreading obtained within 3 minutes and, in the cases of reversibleadhesion, clearing of the detector surface within 10 minutes.Experiments were conducted in a standard rectangular flow cell (1×3×0.05cm), with a long inlet tube (large fluid hold up) and a standardperistaltic pump. Microfluidic techniques can reduce the volume to 0.2ml or less. More dilute solutions can readily be detected but requirelonger times, according to a surface rate law, for instancetransport-limited or first order kinetics which are proportional to thebulk concentration. Regarding sensitivity, near-Brewster opticalreflectometry, was found to reliably detect of adhesion of silicaparticles (200-1000 nm) at surface concentrations as low as 1-micronparticle per 250 um² of area, in water. Fluorescence based detection,for instance TIRF, depends on the density of fluorescent labels on theobject or molecule studied. With 460 nm fluorescent core plain-shellsilica particles, fluorescent signal was reliably obtained at and below1 mg/m², corresponding to 1 particle every 110 um². A similarfluorescence signal with larger particles would correspond to one 1-μmparticle every 1000 μm².

Example 9

Both the size and charge density within positive domains, and the chargedensity on the main negative surface can be adjusted to tune particlesize- and surface-based selectivity. The cationic domains can be flat,in contrast with a second type of patch based on block copolymers. Herean adhesive tail can extend from the flat part of the domain, with theadhesion groups distributed differently along the tail, and with thepotential of accommodating a polypeptide on the chain end. This adhesivetail can provide elasticity and extend the range of attraction duringpull-off. Based on adhesion theory, it provides a means of controllingthe dynamic signature of particles. In all cases, surface domain densitycan be varied through deposition methods while the domain size followsfrom polymer molecular weight.

Example 10

In addition to positive domains made from p-DMAEMA, various other domaincompositions can be derived using known synthetic methods to producepolymers with a variety of structural chemical-physical traits,including the robustness needed for operation in a variety of aqueous ordry environments, and long term stability. As would be understood bythose skilled in the art, a number of known synthetic procedures can beused to prepare homo-, hetero- or copolymeric materials comprising awide variety of functional groups, over a wide range of molecularweights and polydispersities, as may be desired for use in conjunctionwith a particular substrate surface and/or end use application.

Example 11

Adsorption experiments were conducted using a TIRF (total internalreflectance fluorescence) setup built inside a Spex Fluorolog IIFluorescence Spectrometer. This differs from a laser-based instrument inthat the SPEX employs a Xenon lamp with a single monochromator toprovide the excitation light at a chosen wavelength (488 nm), uses adouble monochromator to sort the emissions, and mounts the flow cellvertically instead of horizontally. Fluorescein emissions were measuredat 519 nm.

Example 12

The slit shear flow cell, used in TIRF and reflectometry studies, butalso employed here for controlled protein deposition, in some caseswithout optical monitoring, continually replenishes the bulk solutionand maintains a constant bulk protein concentration. The cell was madeof a Teflon block into which a 1.3 mm deep channel was machined, per amodification of Shibata's design. A microscope slide (or precut siliconwafer or polished glass), comprising the substrate, was then clampedagainst the Teflon surface, and a peripheral O-ring was used to preventleaks. The calibration to convert fluorescence to the adsorbed amount ofprotein identifies regions of transport-limited protein adsorptionkinetics and employs known values for the free solution proteindiffusion coefficients.

Example 13

Once the flow cell was assembled, phosphate buffer solution was passedthrough it for 5 min at a wall shear rate of 24 s⁻¹. The phosphatebuffer solution was switched over to a solution of phosphate bufferedfibrinogen for a set amount of time (depending on the experiment) beforebeing switched back to buffer for 5 min. When protein adsorption wasmonitored via TIRF, this procedure established the fluorescence baselineprior to protein adsorption, and the buffer flush clarified anycontributions of free protein to the signal, as the evanescentpenetration depth of 100 nm is typically greater than the adsorbed layerthicknesses.

Example 14

As described in the literature (Toscano and Santore, Langmuir, supra.)such a flow cell apparatus can be employed to controllably depositprotein on such a surface and confirm coverage. As previously described,solution concentrations can be correlated with surface coverage; proteindeposition is in proportion to protein solution flow time and bulksolution concentration. Studies also suggested little, if any,fibrinogen mobility once deposited.

As discussed above and illustrated below, monodisperse silica particlescan serve as an analyte, to exemplify the workings of the invention.Microscopy, optical reflectometry and various other detection methods ofthe sort discussed above can be used to sense silica particlesinteractive with a heterogeneous surface. Synthetic techniques employedare described more fully in the preceding examples.

Example 15

The impact of flow rate on particle capture on silica surfacescontaining different amounts of fibrinogen was examined. Refer to FIG.16 for 1 μm silica particles. Three different flow rates are compared. Arelevant way to describe the flow rates is in terms of the wall shearrate rather than volumetric flow rate or an average velocity. Much ofthe physics happens at the interface between the fluid and thecollecting surface. By comparison other aspects of the flow which occurfar from the surface are irrelevant. (See, Goldman, A. J.; Cox, R. G.;Brenner, H. Chem. Eng. Sci. 1967, 22, 653-660; describing the force on aspherical particle near a planar wall in shear flow primarily in termsof particle size and the wall shear rate.)

As shown in FIG. 16, the effect of flow can both aid and hinder particlecapture, depending on the surface. For surfaces which have substantialamounts of fibrinogen such that the particle capture rate is near thetransport-limited maxima (to the right of the plots where the datatraces tend to be flat) the particle capture rate increases as the flowis increased. This occurs because when the attraction between particlesand the collecting surface is strong and the fundamental capture rapid,increased flow increases the transport of particles to the interfacialregion, where they adhere immediately. At the opposite extreme, near theadhesion thresholds, the net surface-particle interactions tend to beweak and flow tends to pull particles off the surface. Indeed thehydrodynamic force on particles near a surface in shear flow isproportional to the wall shear rate and the square of the particle size.As the flow is increased, greater surface attractions are needed tocapture particles. Thus the adhesion threshold is shifted to the rightin the direction of requiring greater surface densities of fibrinogen tocapture particles at faster flow rates.

In FIG. 16, it is interesting to note that for some collecting surfacecompositions (around 1.5-1.7 mg/m² fibrinogen, for this particular ionicstrength) as one increases the flow rate, the particle capture ratefirst increases and then decreases—for the reasons described above. Thiseffect is measureable and an indicator that hydrodynamics can beadjusted to either distinguish between different potential analytes orseparate them in tunable ways.

Example 16

Particle adhesion and rolling on different heterogeneous silica surfaceswas examined. These data are shown in FIG. 17, which compares the motionof 1 μm silica particles on pDMAEMA-containing surfaces and onfibrinogen containing surfaces, at an ionic strength of 26 mM.Representative data was obtained using surfaces saturated withfibrinogen, with coverages near 4.5 mg/m². The pDMAEMA containingsurfaces in FIG. 17 were also saturated, with coverages of about 0.45mg/m². Despite the lower coverage of pMDAMEA, this surface is netpositively charged while the fibrinogen saturated surface is netnegative, per FIG. 14.

FIG. 17 provides two data sets at each of 3 flow rates. Each data setshows how many particles are moving at a particular velocity near acapturing surface. Only moving particles are considered here because thedistribution of moving particles reaches a steady state in the flowcell, independent of how long the experiment has been running, whilearrested particles would accumulate with time, per FIGS. 15 and 16.Those which have arrested are not counted in FIG. 17. When particles arenot in intimate contact with the surface, they tend to slip. Whenengaged with the surface, they either roll or arrest. The ability todistinguish between slipping and rolling derives from a fluid mechanicsanalysis (Goldman, supra), which identifies a cut-off velocity, abovewhich rolling cannot occur, and below which rolling must be occurring(at least intermittently). These maximum rolling velocities areindicated for each of the 3 flow rates on the graphs.

In FIG. 17, there are at least 2000 and sometimes up to 10,000 particlesanalyzed for each curve. FIG. 17 reveals that, at each of the 3 flowrates considered, there are small rolling populations when particlescontact any of the surfaces. This rolling population is slightly largeron the fibrinogen coated surface than on the pDMAEMA surface.Accordingly, these data demonstrate that we can indeed find and quantifypopulations of particles that roll on our surfaces and that we can makesmall distinctions in their rolling velocities, depending on surfacechemistry.

Example 17

The data of this example demonstrate interactions of bacteria withheterogenous surfaces of this invention, as can be employed for a numberof end-use applications. The capture rates of staphylococcus aureus(ATCC 25923) was compared with that of 1 μm silica particles on silicasurfaces that are saturated with fibrinogen. As S. aureus is a nearly 1μm sphere resembling silica and also possesses a strongly negative zetapotential, the similar capture rates in FIG. 18 are expected: With rapidintrinsic binding of silica particles or bacteria to fibrinogen, theparticle capture will be transport limited. The slight differences thatseem to be evolving at higher flow rates may be a result of the softernature of the bacteria. Such results illustrate the similarity betweenbacteria and silica particle behavior with respect to heterogenoussurfaces of this invention. Such results also illustrate use of thepresent invention to sense and/or detect bacteria and other biologicalanalytes and confirm the utility of silica particles as a model forsensing various other particles or analytes.

1. A system for selective particle sensing, said system comprising: aheterogeneous surface comprising a negatively-charged surface member anda plurality of components thereon, said components spaced about saidsurface member and having a density thereon, each said componentcomprising a net charge; and a fluid medium comprising a suspendedparticle comprising a net charge, said medium contacting saidheterogeneous surface.
 2. The system of claim 1 wherein said surfacemember comprises silica.
 3. The system of claim 1 wherein said spacedcomponents comprise a protein.
 4. The system of claim 3 wherein saidspaced protein components comprise a fibrinogen.
 5. The system of claim1 wherein said spaced components comprise less than about 50% of thesaturation level of said heterogeneous surface, said protein componentsnanometrically spaced.
 6. The system of claim 5 wherein said particle ismicron-dimensioned.
 7. The system of claim 6 wherein the zone ofinfluence of said particle has a radius at least about the averagedimension between said spaced components.
 8. A system for selectiveparticle separation, said system comprising: a heterogeneous surfacecomprising a negatively-charged surface member and a plurality ofnanodimensioned protein components thereon, said protein componentsspaced about said surface member and having a density thereon, each saidcomponent comprising a net charge; and a fluid medium comprising amixture of particles, at least one said particle comprising a netcharge, said medium contacting said heterogeneous surface.
 9. The systemof claim 8 wherein said surface member comprises silica.
 10. The systemof claim 8 wherein said spaced protein components comprise a fibrinogen.11. The system of claim 8 wherein said spaced components comprise lessthan about 50% of the saturation level of said heterogeneous surface,said protein components nanometrically spaced.
 12. The system of claim11 wherein said particle is micron-dimensioned.
 13. The system of claim12 wherein the zone of influence of said particle has a radius at leastabout the average dimension between said spaced components.
 14. A systemfor selective particle sensing, said system comprising: a heterogeneoussurface comprising a negatively-charged surface member and a pluralityof nanodimensioned protein components thereon, said protein componentsnanometrically-spaced about said surface member and comprising less thanabout 50% of the saturation level of said heterogeneous surface, eachsaid component comprising a net charge; and a fluid medium comprising asuspended particle comprising a net charge, said medium contacting saidheterogeneous surface.
 15. The system of claim 14 wherein said surfacemember comprises silica.
 16. The method of claim 14 wherein said proteincomponents are spaced an average distance, said distance ranging fromabout 15 nanometers to about 60 nanometers, from about component centerto about component center.
 17. The system of claim 16 wherein saidspaced protein components comprise a fibrinogen.
 18. The system of claim17 wherein each said spaced component has a dimension up to about 50nanometers.
 19. The system of claim 14 wherein said particle ismicron-dimensioned.
 20. The system of claim 19 wherein the zone ofinfluence of said particle has a radius at least about the averagedimension between said spaced components.